The development of drug-resistant strains of B. anthracis is technically quite feasible, and could be a substantial threat in future terrorist attacks. Building on a well-established collaborative network amongst the investigators participating in this project, we propose an integrated approach toward the development of new antimicrobials, new potentiators of existing antimicrobials, and direct inhibitors of the anthrax toxin as strategies to combat natural and bioengineered forms of B. anthracis. We will use a combination of strategies, beginning with genetic identification and validation of novel bacterial targets, determination of target 3D molecular structures, utilization of diverse chemical libraries for high throughput screening, structure-based drug design, synthesis of lead compounds and their optimization, followed by macrophage and animal testing. An important strength of the application is the broad range of the participants' expertise, including bacterial genetics and biochemistry, structural biology of macromolecules, computer-assisted drug design, synthetic chemistry, macrophage biology, animal modeling and clinical infectious disease. Project 1 will identify and validate new antibiotic targets in ribosomal RNA. Project 2 will identify and validate new infection-related targets in Bacillus anthracis. Project 3 will utilize structure-based design and high throughput screening to develop lead inhibitors of currently known and to-be-identified antibiotic targets. Project 4 will develop inhibitors to prevent the binding of the B. anthracis toxin to the cellular receptor. Project 5 will evaluate the role of antibiotics in modulating cytokine activation and toxin triggering following macrophage infection and animal model development. Four scientific cores will support these projects: A protein expression core will provide proteins for target evaluation and structure-based design. A macromolecular characterization and structure core will provide structural and thermodynamic information for target characterization and structure-based design. A chemical improvement core will provide synthetic design and optimization of lead inhibitors. A bioassay core will provide a variety of assays for identifying and evaluating lead therapeutic agents. An administrative core will provide fiscal management and administrative support.